We previously reported that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) and 4'-C-cyano-2'-deoxyguanosine (CdG) highly potently suppressed the replication of wild-type HBV strains (HBVWT) as examined in HepG2.2.2.15.7 cells, HBVWT-plasmid-transfected Huh7 cells, and in HBVWT-exposed human-liver-chimeric-uPA+/+/SCID+/+ (hu-liver-chimeric) mice. CAdA and CdG were also potent against a highly entecavir (ETV)-resistant HBV variant containing three critical amino acid substitutions: L180M, S202G, and M204V (HBVL180M/S202G/ M204V) as examined in HBVL180M/S202G/M204V-plasmid-transfected Huh7 cells and HBVL180M/S202G/M204V-exposed hu-liver-chimeric mice. However, both compounds proved to be greatly more cytotoxic than ETV. We thus continued the design and synthesis of approximately 220 novel nucleoside analogs containing a cyano moiety at the 4'-position of the ribose and identified 3'-C-cyano-2-amino-9-[(1S,3R,4S)-4'-hydroxy-3'- hydroxymethyl]-2'-methylidene-cyclopentyl]-1H-purin-6-one (CMCP). It is of note that CMCP structurally resembles CdG in that both compounds have 4'-cyano moiety and also resembles ETV in that both of CMCP and ETV have an exocyclic double bond or methylidene in the 4' position of the cyclopentyl moiety. To determine whether CMCP reduces the amount of intracellular HBVWT DNA in HepG2.2.2.15.7 cells, DNA isolated from HepG2.2.2.15.7 cells, which were cultured in the presence of CMCP over 14 days, was subjected to real-time HBV-PCR as described in the Materials & Methods. Antiviral activity was scored as the percent inhibition relative to that of drug-unexposed control cells. As shown in Table 1, the activity of CMCP against HBVWT was significantly more potent than ADV and TDF and slightly more potent than 3TC and TAF, although that of CMCP was significantly less potent than that of ETV or CdG. Representative titration curves comprised of the data from three independent assays giving 50% inhibitory concentrations (IC50s) of 1.7, 0.5, and 23 nM to block HBV DNA synthesis in HepG2.2.2.15.7 cells for ETV, CdG, and CMCP, respectively. We further examined the effects of CMCP on cellular mitochondrial DNA (mtDNA), cell growth, and cell viability using MOLT-4 and Daudi. After 7 days of culture in the presence of various concentrations of a test agent, the cells were harvested and subjected to determination of cell growth, cell viability and DNA extraction (for real-time mtDNA-PCR). In the case of ETV, a sharp decline in cell viability and growth was seen at 10 microM in MOLT-4 cells and the same was seen at 100 microM in Daudi cells. There was also a sharp reudction in the amount of mtDNA at 10 and 100 microM in MOLT-4 and Daudi cells, respectively. By contrast, there was no sharp reduction when the cells were cultivated in the presence of CMCP in neither of MOLT or Daudi cells. There was only a slight reduction of cell viability, cell growth, and the amount of mtDNA at 100 microM. We subsequently asked whether CMCP blocked the replication of HBVWTCe, ETV-resistant HBV (HBVETV-RL180M/S202G/M204V) and ADV-resistant HBV (HBVADV-RA181T/N236T). ETV effectively reduced the synthesis of HBVWTCe at 10 and 102 nM concentrations giving an IC50 value of 16 nM. However, as expected, ETV at 1 to 105 nM concentrations failed to effectively block the DNA synthesis of HBVETV-RL180M/S202G/M204V even at the highest concentration (105 nM), giving ETV's IC50 value of 64,040 nM, although ETV fairly well blocked the synthesis of HBVADV-RA181T/N236T DNA, giving an IC50 value of 105. ADV moderately well blocked the synthesis of HBVWTCe and HBVETV-RL180M/S202G/M204V DNA, giving IC50 values of 1,163 and 30,770, respectively, while it failed to block the synthesis of HBVADV-RA181T/N236T DNA, giving an IC50 value of 120,870. TDF quite well suppressed the synthesis of HBVWTCe, HBVETV-RL180M/S202G/M204V, and HBVADV-RA181T/N236T, giving IC50 values of 177, 406, and 218, respectively. CMCP also effectively blocked the synthesis of HBVWTCe and HBVADV-RA181T/N236T DNA with the IC50 values of 206 and 96 nM, respectively, and it fairly well blocked the DNA synthesis of HBVETV-RL180M/S202G/M204V, giving an IC50 value of 2,657. We then asked whether CMCP blocked the replication of HBV in hu-liver-chimeric mice. In 8 weeks following the inoculation of such mice with HBVWTCe, they were orally gavaged with ETV or CMCP (prepared at a concentration of 0.1 mg/mL in saline) using oral sondes so that the dose administered resulted in 1 mg/kg/day. Just before the administration of ETV, HBV copy numbers in their plasma were as high as 9 x 108/ml; however, the viremia levels significantly went down by day 7 of ETV administration and further viremia reduction occurred even after the termination of ETV administration to the lowest (by day 21). CMCP comparably blocked the replication of HBVWTCe under the same conditions (p=0.10). In 8 weeks following the inoculation of hu-liver-chimeric mice with an HBVETV-RL180M/S202G/M204V, the viremia levels had reached 7 x 107 to 2 x 108/ml. However, ETV, at a dose of 1 mg/kg/day, showed essentially no reduction in the HBVETV-RL180M/S202G/M204V viremia levels. By contrast, CMCP at the same dose of 1 mg/kg/day, brought about a significant level of viremia reduction by day 7 of administration. The greatest magnitude of viremia reduction with CMCP was -1.1 log10 copies/ml (average of -1.2 and -1.0 log 10). Over the 14-day period of administration, the viremia reduction in CMCP-receiving mice was significantly lower compared to that in ETV-receiving mice (p0.0001). We finally analyzed the structural interactions of ETV-TP and CMCP-TP with reverse transcriptase (RT) of HBVWT. There are multiple strong polar interactions between CMCP-TP and the reverse transcriptase. The amino substituent of the purine forms a hydrogen bond interaction with the backbone carbonyl of Met171. The 4-hydroxy substituent of the cyclopentyl group forms a hydrogen bond with the backbone nitrogen of Phe88. The triphosphate group of CMCP-TP forms polar interactions with multiple amino acid residues of the RT, Ser85, Ala86, and Ala87. The triphosphate group interacts with a magnesium ion, which also interacts with Asp83 and Val84. These networks of strong polar interactions must be responsible for stabilizing the binding of CMCP-TP in the active site of RT of HBVWT. Both CMCP-TP and ETV-TP form good van der Waals interactions with Met204 and Asp205 in the active site of HBVWT RT as seen from the analysis of the Connolly surface interactions. Importantly, the 4'-cyano group that CMCP contains in its cyclopentyl group forms good van der Waals interactions with Leu180 of RT of HBVWT. This Leu180 interaction is also present in the complex of RT of HBVETV-RL180M/S202G/M204V. However, there is a distinct difference in the non-polar interactions of ETV-TP with the RT of HBVETV-RL180M/S202G/M204V. While the methylidene group maintains some nonpolar interactions with the substituted Val204, the interaction with the substituted Met180 is completely lost for ETV-TP complexed with the RT of HBVETV-RL180M/S202G/M204V. This is because of the lack of the 4'-cyano group in ETV compared to CMCP. Thus, from the observation that the interactions of the RT of HBVETV-RL180M/S202G/M204V with M204V and D205 persist in both complexes of CMCP-TP and ETV-TP with the RT of HBVETV-RL180M/S202G/M204V, it is most likely that the difference in the interactions of CMCP-TP over ETV-TP with the RT of HBVETV-RL180M/S202G/M204V arises because of the sustained interactions of the cyano group of CMCP-TP with Leu180 in the RT of HBVWT as well as with the substituted amino acid Met180 in the RT of HBVETV-RL180M/S202G/M204V. We are presently focusing on CFCP, another nucleoside reverse transcriptase inhibitor, which is more potent against HBV than CMCP.